Figure S5.
Early release of RUSH-α5 is sensitive to GRASP silencing. (A) Immunoblot of GST pull-downs of recombinant integrin α5 WT or mutant (ΔSDA or PPAA) peptides incubated with GST alone or recombinant GST-GRASP65. No enrichment of GST-GRASP65 signal over background (no peptide beads) is detected with integrin α5 WT peptide, indicating that the integrin α5 peptides do not interact with recombinant purified GST-GRASP65. (B) ELISA assay detecting biotinylated recombinant integrin α5 WT or ΔSDA or PPAA mutant with HRP-streptavidin incubated on wells coated with GST alone or GST-GRASP65. No direct interaction between GRASP65 and integrin α5 WT peptide was detected. (C) Immunoblot of lysates collected from control-silenced or GRASP65 and GRASP55-silenced U2OS cells used in D, E, probed for GRASP65 and GRASP55. β-actin was probed as a loading control. (D) Representative immunofluorescence images of control-silenced or GRASP65 and GRASP55-silenced U2OS cells expressing RUSH-α5 and plated on dual-coated micropatterns (magenta dots, FN; non-fluorescent regions, collagen peptide GFOGER). (E) Relative recruitment of RUSH-α5 in control- or GRASP65- and GRASP55-silenced U2OS cells to FN dots within the cell boundary. Data are mean ± SD; n = 9 siCTRL cells, 11 siGRASP cells (36 and 44 dots, respectively) from one experiment. (F and G) Quantification of RUSH-α5 intensity in the four areas relative to signal intensity in the respective area 2.5 min prior to RUSH-α5 appearance in (F) new adhesions or (G) already existing adhesions (determined from the time-lapse images) on FN- and anti-GFP antibody-coated surfaces. Adhesions close to the cell edge and with a minimum lifetime of 15 min were analyzed and changes of RUSH-α5 intensity were plotted over time in the indicated areas ranging from distal to proximal to the cell body. Data are mean ± SD; One independent experiment 9 adhesions from 6 cells on 2 coverslips (F) and one independent experiment 9 adhesions from 5 cells on 2 coverslips (G). (H and I) Representative images (H) and (I) track maps related to Fig. 6 I. Red arrows indicate direction of adhesion growth. Scale bars: 20 µm. Source data are available for this figure: SourceData FS5.

Early release of RUSH-α5 is sensitive to GRASP silencing. (A) Immunoblot of GST pull-downs of recombinant integrin α5 WT or mutant (ΔSDA or PPAA) peptides incubated with GST alone or recombinant GST-GRASP65. No enrichment of GST-GRASP65 signal over background (no peptide beads) is detected with integrin α5 WT peptide, indicating that the integrin α5 peptides do not interact with recombinant purified GST-GRASP65. (B) ELISA assay detecting biotinylated recombinant integrin α5 WT or ΔSDA or PPAA mutant with HRP-streptavidin incubated on wells coated with GST alone or GST-GRASP65. No direct interaction between GRASP65 and integrin α5 WT peptide was detected. (C) Immunoblot of lysates collected from control-silenced or GRASP65 and GRASP55-silenced U2OS cells used in D, E, probed for GRASP65 and GRASP55. β-actin was probed as a loading control. (D) Representative immunofluorescence images of control-silenced or GRASP65 and GRASP55-silenced U2OS cells expressing RUSH-α5 and plated on dual-coated micropatterns (magenta dots, FN; non-fluorescent regions, collagen peptide GFOGER). (E) Relative recruitment of RUSH-α5 in control- or GRASP65- and GRASP55-silenced U2OS cells to FN dots within the cell boundary. Data are mean ± SD; n = 9 siCTRL cells, 11 siGRASP cells (36 and 44 dots, respectively) from one experiment. (F and G) Quantification of RUSH-α5 intensity in the four areas relative to signal intensity in the respective area 2.5 min prior to RUSH-α5 appearance in (F) new adhesions or (G) already existing adhesions (determined from the time-lapse images) on FN- and anti-GFP antibody-coated surfaces. Adhesions close to the cell edge and with a minimum lifetime of 15 min were analyzed and changes of RUSH-α5 intensity were plotted over time in the indicated areas ranging from distal to proximal to the cell body. Data are mean ± SD; One independent experiment 9 adhesions from 6 cells on 2 coverslips (F) and one independent experiment 9 adhesions from 5 cells on 2 coverslips (G). (H and I) Representative images (H) and (I) track maps related to Fig. 6 I. Red arrows indicate direction of adhesion growth. Scale bars: 20 µm. Source data are available for this figure: SourceData FS5.

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